Hydrogen sulfide (H2S), which is well known as a toxic gas, is produced endogenously in mammalian tissues from L-cysteine mainly by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine beta-synthetase and cystathionine gamma-lyase. Recently, we showed that cystathionine beta-synthetase in the brain produces H2S, and that H2S facilitates the induction of hippocampal long-term potentiation by enhancing NMDA receptor activity. Here we show that mRNA for another H2S producing enzyme, cystathionine gamma-lyase, is expressed in the ileum, portal vein, and thoracic aorta. The ileum also expresses cystathionine beta-synthetase mRNA. These tissues produce H2S, and this production is blocked by cystathionine beta-synthetase and cystathionine gamma-lyase specific inhibitors. Although exogenously applied H2S alone relaxed these smooth muscles, much lower concentrations of H2S greatly enhanced the smooth muscle relaxation induced by NO in the thoracic aorta. These observations suggest that the endogenous H2S may regulate smooth muscle tone in synergy with NO.